Interaction of zonula occludens-1 (ZO-1) with α-actinin-4: Application of functional proteomics for identification of PDZ domain-associated proteins

Vincent C. Chen, Xinbo Li, Hélène Perreault, James I. Nagy

Research output: Contribution to journalArticlepeer-review

32 Scopus citations

Abstract

The use of recombinant "bait" proteins to capture protein-binding partners, followed by identification of protein interaction networks by mass spectrometry (MS), has gained popularity and widespread acceptance. We have developed an approach using recombinant PDZ protein interaction modules of the membrane-associated guanylate kinase (MAGUK) protein zonula occludens-1 (ZO-1) to pull-down and screen for proteins that interact with these modules via their PDZ domain binding motifs. Identification of proteins by MS of pull-down material was achieved using a vacuum-based chromatography sample preparation device designed for matrix-assisted laser desorption/ionization (MALDI) MS. MS analysis of tryptic fragments in pull-down material revealed a number of potential ZO-1 interacting candidates, including the presence of peptides corresponding to the cortical membrane scaffolding protein α-actinin-4. Interaction of α-actinin-4 with ZO-1 was confirmed by coimmunoprecipitation of these two proteins from cultured cells, as well as from brain, liver, and heart, and by immunoblot detection of α-actinin-4 after pull-down with the first PDZ domain of ZO-1. In contrast, the highly homologous α-actinin family member, α-actinin-1, displayed no association with ZO-1. Immunofluorescence showed colocalization of α-actinin-4 with ZO-1 in cultured HeLa and C6 glioma cells, as well as in a variety of tissues in vivo, including brain, heart, liver, and lung. This study demonstrates the utility of MS-based functional proteomics for identifying cellular components of the ZO-1 scaffolding network. Our finding of the interaction of ZO-1 with α-actinin-4 provides a mechanism for linking the known protein recruitment and signaling activities of ZO-1 with α-actinin-4-associated plasma membrane proteins that have regulatory activities at cell-cell and cell-extracellular matrix contacts.

Original languageEnglish (US)
Pages (from-to)2123-2134
Number of pages12
JournalJournal of Proteome Research
Volume5
Issue number9
DOIs
StatePublished - Sep 2006
Externally publishedYes

Keywords

  • Functional proteomics
  • MAGUK
  • MALDI
  • MS
  • PDZ
  • Vacuum chromatography
  • ZO-1
  • α-actinin-1
  • α-actinin-4

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

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